CN101983067B - The treatment of microorganism infection - Google Patents
The treatment of microorganism infection Download PDFInfo
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- CN101983067B CN101983067B CN200980112527.0A CN200980112527A CN101983067B CN 101983067 B CN101983067 B CN 101983067B CN 200980112527 A CN200980112527 A CN 200980112527A CN 101983067 B CN101983067 B CN 101983067B
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
- C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/085—Staphylococcus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1271—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Micrococcaceae (F), e.g. Staphylococcus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
Abstract
The present invention relates to improved microbial antigen vaccine, pharmaceutical composition, immunogenic composition and antibody and its purposes in treatment microorganism infection particularly bacterial origin includes the microorganism infection in staphylococcus source.It is desirable that the present invention relates to be used for restructuring staphylococcus MSCRAMM or MSCRAMM sample albumen or its fragment that the combination to its host's part for the treatment of is reduced.
Description
Foreword
The present invention relates to improved microbial antigen vaccine, pharmaceutical composition, immunogenic composition and antibody and its
Treatment microorganism infection, particularly bacterial origin, including the microorganism infection that staphylococcus (Staphylococcal) is originated
In purposes.
Multi-drug resistant (MDR) is increasingly serious problem of the gram-positive bacterium especially in hospital.Antibiotic and
Widely using for the reagent of other treatment bacterium infections has caused bacterium to produce the resistance for the reagent, Hen Duoxi rapidly
Bacterium has multi-drug resistant.Therefore, need to provide the treatment of resistant infections as improved treatment at present.
Staphylococcus is spherical gram-positive bacterium, and it is generally arranged according to the irregular cluster of grape sample.Have
The member of the normal flora (flora) of human skin and mucous membrane, it is other, cause ecpyesis formed, it is various suppurative
Infection, even fatal septicaemia.Pathogenic staphylococcus generally trigger haemolysis, make the clotting of plasma and produce outside various kinds of cell
Enzyme and toxin.
There are at least 30 kinds in staphylococcus.Three essentialspecies with clinical importance are staphylococcus aureuses
(Staphylococcus aureus), MRSE (Staphylococcus epidermidis) and saprophytic grape ball
Bacterium (Staphylococcus saprophyticus).Staphylococcus aureus is coagulase positive, this by its with it is other
Plant and distinguish.Staphylococcus aureus is the main pathogens for the mankind.Almost everyone has in life at it
The infection of staphylococcus aureus of type, from the seriousness or minor skin infections of food poisoning to the sense of serious life-threatening
Dye.The staphylococcus of coagulase-negative is normal mankind's flora, and it causes infection sometimes, generally related to transplantation device, special
It is not in very young, old and immune deficiency patient.About 75% is drawn by the staphylococcus of coagulase-negative
The infection for rising all is caused by MRSE.By walsh staphylococcus (Staphylococcus warneri), people Portugal
The infection that grape coccus (Staphylococcus hominis) and other kinds cause is less common.Staphylococcus saprophyticus is young woman
The relatively common inducement of urinary tract infection in female.Staphylococcus produces catalase, and this is by itself and streptococcus
(streptococci) it is distinguished.Road Deng staphylococcus (S.lugdunensis) is also clinically relevant, and it is present in about
In the case of the infectious endocarditis of 5-10%.
In joint space, staphylococcus aureus is in main component for colonizing in the articular cartilage of collagen seemingly promotees
Into the key factor that septic arthritis is produced.The bacterial arthritis that hematopoietic is obtained is still serious medical problem.This
The arthropathy for planting rapid progression and high-destruction is difficult to eradicate.Typically, do not produce if serious joint injury, have
Less than 50% infected patient is difficult to recover.Staphylococcus aureus is suffered from from hematopoietic and the scorching adult of secondary bone marrow
The main pathogens for being separated in person and being come.
In inpatient, staphylococcus such as staphylococcus aureus is main infection inducement.Wound or indwelling
The initial local infection of medicine equipment can result in and be more heavily invasive sexy dye such as septicaemia, osteomyelitis, mastitis and the heart
Intimitis.In the infection relevant with medicine equipment, after this in the short period plastics and metal surface by host plasma
It is coated with stromatin such as fibrinogen and fibronectin.Staphylococcus aureus and other staphylococcuses adhere to these
This ability on albumen is initially essential for what is infected.Blood vessel graft, intravenous catheter, artery heart valve and
Heart-assist device is thrombotic, it is easy to the cluster of bacterium.In general, in staphylococcus, Staphylococcus aureus
Bacterium is most destructive pathogen in these infection.
Had been observed that in hospital worldwide to being currently available that most antibiotic for the treatment of infection are presented
The staphylococcus aureus chorista of resistance is dramatically increased.The exploitation for resisting the penicillin of staphylococcus aureus is infection control
A much progress in system and treatment.Unfortunately, the biology of penicillin resistance is rapid occurs, in the urgent need to new antibiotic.With
The introduction of every kind of new antibiotic, staphylococcus aureus can be to anti-beta-lactamase --- the penicillin combination egg of change
In vain, and allow bacterium exist mutation epicyte protein.Therefore, the golden yellow grape of Methicillin resistance is occurred in that
The biology of coccus (MRSA) and multidrug resistant and global hospital and sanatorium leave main footprint (Chambers,
H.F., Clin Microbiol Rev, 1：173,1988；And Mulligan, M.E. et al., AmJ Med, 94：313,1993).
At present, causing in the aureus strains of the infection in hospital almost has half for all antibiosis in addition to vancomycin
Element is all resistance, it appears that vancomycin become it is invalid also be the time problem.
Therefore, the urgent and increasingly desirable therapeutic agent for treating staphylococcus such as infection of staphylococcus aureus, institute
State the bacterium bacterial strain that therapeutic agent is effective against those antibiotic resistances.
In gram positive pathogens such as staphylococcus, streptococcus and enterococcus, it is referred to as adhesin (adhesin)
Albumen for example, by promoting cluster, be attached to blood clotting and make tissue damaged and mediate such infection.These specificity
Antimicrobial surface adhesin be referred to as MSCRAMM (microbial surface componenets identity adhesive matrix molecule) (Patti, J. et al.,
Ann Rev Microbiol, 48：585-617,1994；Patti, J.and Hook, M., Cur Opin Cell Biol., 6：
752-758,1994).MSCRAMM specific recognitions are simultaneously bound to extracellular matrix (ECM) composition, such as fibronectin, fiber
Proteinogen, collagen and elastin laminin.These MSCRAMM are found that in many gram positive pathogens, its amino acid sequence
It is related, they have similar module (modular) design and common binding structural domain group structure.
The part in MSCRAMM and host tissue on bacterial cell surface is interacted with key pattern, so as to cause
Bacterial adhesion is to host.Bacteria live generally needs adhesion, and it helps bacterium to evade the defense mechanism of host and choosing for antibiotic
War.Once bacterium successfully adheres to and is clustered in host tissue, its physiology is to be changed significantly, and secretes destructive composition example
Such as toxin and enzyme.Additionally, Adherent bacteria generally produces biomembrane and the rapid killing effect to most antibiotic produces resistance.
Bacterium can express the MSCRAMM for recognizing various stromatins.Ligand binding site in MSCRAMM seemingly by
What the relatively short continuous section (motif) of amino acid sequence was determined.Because being can be found that in several different bacterial species
Similar motif, so these functional motifs seem that inter-species transfer (Patti and Hook, Cur Opin Cell can be carried out
Biol, 6：752-758,1994).In addition, sometimes single MSCRAMM can combine several ECM parts.
MSCRAMM can be situated between by with the protein binding including fibrinogen (Fg) and/or fibronectin (Fn) etc.
Lead infection.Fibrinogen and fibronectin are the albumen found in blood plasma, and it plays a significant role in hemostasis and solidification.
6 polypeptide chain compositions of fibrin reason：2 A α, 2 B β and 2 γ-chains.The C-terminal part of γ-chain is in life
It is important in thing, and is interacted with platelet integrin in platelet adhesion and aggregation.The region is also gold
What staphylococcus aureus were targeted, cause cell aggregation and the tissue adsorptions of fibrinogen dependence.
Staphylococcus aureus has the albumen of several surface expressions, and its stimulating platelet is activated and assembled.Golden yellow Portugal
The MSCRAMM albumen of grape coccus includes but is not limited to following albumen：
- fibrinogen binding protein clumping factor A (ClfA)；
- fibrinogen binding protein clumping factor B (ClfB)；
- fibronectin-fibrinogen binding protein A (FnBPA)；
- fibronectin-fibrinogen binding protein B (FnBPB)；With
- staphylococcus aureus surface Protein S asA, SasG, SasK etc..
Table 1 below lists the selected works of various aureus cell wall anchoring surface albumen
Table 1
aAa, the length protein represented with amino acid.
bThe molecular chaperones that albumen is recognized and combined.
cConsensus motif that is that sorting enzyme is recognized and being present in C-terminal cell membrane sorting signals.
dSorting enzyme with cell wall surface proteins as substrate.
eTNFR：Tumor Necrosis Factor Receptors.
fAlso with the epithelial cell for coming off in protein binding.Promote to sterilized lipid and the resistance of lactoferrin.
gAlso combined with the nasal epithelial cells for coming off.Participate in the formation of biomembrane.
Egg of other staphylococcus expression similar to clumping factor or protein-bonded surface expression listed above
In vain (MSCRAMM).These are included but is not limited to：
- SdrF, SdrG and SdrH from MRSE, wherein having shown that SdrG/F and fibrinogen and glue
Original is combined.
- it is the albumen combined with fibrinogen from the staphylococcic Fbl of road Deng.Fbl is the member of Sdr families, Sdr
Family is one group contains the aureus cell surface protein of characteristic serine-aspartate duplicate block.Fbl is mapped out
Fibrinogen binding structural domain be 313 amino acid, and with the clumping factor A (ClfA) from staphylococcus aureus
Respective regions have 62% homogeneity.
Other albumen/adhesins with ligand binding include Isd albumen (the surface determinant of ion regulation), although it
Be not all MSCRAMM (such as IsdB and IsdH) in itself, but they promote bacterial adhesions to extracellular matrix components,
Herein referred to as " MSCRAMM samples albumen ".Know that IsdA promotes the adhesion to squamous cell, and for fibre
Former and fibronectin the compatibility of fibrillarin is weak, so being technically defined as MSCRAMM.
Clumping factor A (ClfA) is first golden yellow Portugal of the identified γ-chain combination with fibrinogen out
Grape coccus adhesin.Then recognize fibronectin-fibrinogen binding protein A (FnBPA) and fibronectin-fibrin
Former associated proteins B (FnBPB) is the bifunctional protein that identical C-terminal peptide section is combined in γ-chain with Fg.ClfA and FnBP
Include the architectural feature that ClfB has with all Cell wall anchored proteins expressed in gram-positive bacteria.
For example, clumping factor A (ClfA) is the albumen on surface of staphylococcus aureus.ClfA is golden yellow Portugal
The important virulence factor of grape coccus.It facilitates septic arthritis and endocarditic pathology to occur.ClfA is with similar
The archetype of the surface associated protein family (including but not limited to ClfB, SdrD, SdrE etc.) of structure/module group structure.
ClfA contains 520 N-terminal A domains (fibrinogen calmodulin binding domain CaM) of amino acid, and it includes three individually
Subdomain N1, N2 and N3 of folding.A domains be followed by serine-aspartate dipeptides repeat region and cell membrane and
Film crosses over region, and it contains the LPDTG motifs to cell wall anchor promoted for sorting enzyme.ClfA actually exists in all
Staphylococcus aureus strains in (Peacock SJ, Moore CE, Justice A, Kantzanou M, Story L,
Mackie K, O ' NeillG, Day NPJ (2002) Virulent combinations of adhesin and toxin
genes in natural populations of Staphylococcus aureus.Infect Immun 70：4987-
4996).It is combined with the C-terminal of the γ-chain of fibrinogen, therefore, it is possible to induce the bacterial accumulation in fibrinogen solution
(McDevitt D, Nanavaty T, House-Pompeo K, Bell E, Turner N, McEntire L, Foster T,M(1997)Characterization of the interaction between the Staphylococcus
aureus clumping factor(ClfA)and fibrinogen.Eur J Biochem247：416-424；With
McDevitt D, Francois P, Vaudaux P, Foster TJ (1994) Molecular characterization of
the clumping factor(fibrinogen receptor)ofStaphylococcus aureus.Mol Microbiol
11：237-248).
The three-dimensional structural analysis that the fibrinogen binding protein SdrG and ClfB of ClfA and correlation are carried out are had revealed that：
In all these GAP-associated protein GAPs, ligand binding A domains are constituted by three subdomains N1, N2 and N3, wherein corresponding to
The residue 221-559 of region N2-N3 is the truncate (truncate) of minimum reservation and fibrinogen binding ability.
It was found that amino acid residue 532 to 538 corresponds to locking peptide (latching peptide) region of ClfA.Each subdomain bag
Containing 9 β chains, it constitutes new IgG types and folds.Fibrinogen γ chains binding site peptide point in these albumen be located at N2 and N3 it
Between node drain tank.It has been found that the three-dimensional structure of these albumen has significant structural similarity, this is due to existing
One or more related amino acid sequences, similar module design and common binding structural domain group structure.
SdrC, SdrD, SdrE, FnBPA-A (all 7 homotypes) and FnBPB-B (all 7 homotypes) have similar mould
Block group structure, therefore use PHYRE molecular models, it is contemplated that these albumen will be with identical three-dimensional structure.
IsdA and IsdB is without the structure with Clf or Sdr albumen same types.They have and new are referred to as NEAT's
Motif, the motif participates in ligand binding.But, NEAT motifs are made up of (by 2 antiparallel β pieces of 5 chains β chain interlayer structures
The sandwich foldings of β of layer composition) and be Ig superfamily members (Pilpa et al. " Solution Structure of the NEAT
(NEAr Transported)Domain from IsdH/HarA：the Human Hemoglobin Receptor in
Staplococcus aureus”J.Mol.Biol.(2006)360：435-447), in this sense, NEAT motifs with
The three-dimensional structure of Clf or Sdr is similar.In having solved the three-dimensional structure of the NEAT motifs of IsdH and predicting ring 1b-2
Residue.
The expression of ClfA hinders the phagocytosis of macrophage and neutrophil cell on staphylococcus aureus
(Palmqvist N, Patti JM, Tarkowski A, Josefsson E (2004) Expression of
staphylococcal clumping factor A impedes macrophage phagocytosis.Microb
Infect 6：188-195；With Higgins J, Loughman A, van Kessel KPM, van Strijp JAG, Foster
TJ(2006)Clumping factor A of Staphylococcus aureus inhibits phagocytosis by
human polymorphonuclear leukocytes.FEMS Microbiol Lett258：290-296).In neutrophil(e) granule
In cell, this is caused jointly by fibrinogen dependent mechanism and fibrinogen dependent/non-dependent mechanism.Conversely, bacterium
The ClfA of expression activates blood platelet by there is interaction with GPIIb/IIIa, so as to cause aggregation.Work as fibrinogen
In the presence of, this effect carries out maximally efficient；But also there are the fibrinogen independent pathways of platelet activation
(Loughman A, Fitzgerald JR, Brennan MP, Higgins J, Downer R, Cox D, FosterTJ (2005)
Roles of fibrinogen, immunoglobulin and complement in platelet activation
promoted by Staphylococcus aureus clumping factor A.Mol Microbiol 57：804-818；
With O ' Brien L, Kerrigan SW, Kaw G., Hogan M., Penad é sJ., Litt D., Fitzgerald D.J.,
Foster T.J.&Cox D.(2002)Multiple mechanisms for the activation of human
platelet aggregation by Staphylococcus aureus：roles for the clumping factors
ClfA and ClfB, the serine-aspartate repeat protein SdrE and protein A.Mol
Microbiol 44,1033-1044).
ClfA is virulence factor (Josefsson E., the Hartford O., O ' of the septic arthritis in inducing mouse
Brien L, Patti JM, Foster T (2001) Protection against experimental Staphylococcus
Aureus arthritis by vaccination with clumping factor A, a novel virulence
determinant.J Infect Dis 184：1572-1580).Additionally, ClfA is together with another fibrinogen binding protein
The elimination of ClfB causes resists protection (Palmqvist N, the Foster T, Fitzgerald of systemic inflammatory in infection early stage
R, Josefsson E, Tarkowski A (2005) Fibronectin-binding proteins and fibrinogen-
binding clumping factors play distinct roles in staphylococcal arthritis and
systemic inflammation.J Inf Dis191：791-798).
Fibrinogen binding protein ClfA that is separated and characterizing staphylococcus aureus, it is such as U.S.
The theme of the patent No. 6,008,341 and 6,177,084.
There is identical structure (three-dimensional) to organize structure and about 27% amino acid identities for ClfA and ClfB.FnBPA with
ClfA has about 25% amino acid identities.
The vaccine based on MSCRAMM there is no to go through and list at present.It is a kind of grape of donor seletion
The intravenous human immunoglobulin of coccus (IGIV), its targeting ClfA and SdrG, due to showing poor in III clinical trial phases,
So being revoked from clinical test.Currently reappraise to determine it whether the feasibility of staphy lococcus infection is treated.
WO 2005/116064 is related to FnBPA, and it is the multi-functional associated proteins of staphylococcus aureus.The N ends of FnBPA
End A domains are similar to ClFA, it has been found that it is combined with fibrinogen.But, C-terminal BCD domains and the fibre of FnBPA
Connect protein binding, therefore, FnBPA is difunctional MSCRAMM.
WO 2005/116064 based on the finding that：In the case where there is TGase, in bacterial adhesion element
Formed between FnBPA and host protein fibronectin and be covalently attached, so that with reference to more strong and substantially irreversible
's.Fibrinogen is the main component (~3mg/ml) in blood, in blood its as coagulation cascade final target.It is fine
Even protein content is relatively low ,~0.3mg/ml or a Fn molecule original for every 10-15 fibrin.Fibrinogen and
Fibronectin is considered as unrelated with blood, and they are independently circulated in blood.
Importantly, the covalent cross-linking of the more particularly to factor XIIIa catalysis of WO 2005/116064.WO2005/116064
The multiple mutant in restructuring FnBPA are separated, wherein the residue (i.e. transglutaminase substrate) with positively charged side chain
It is changed.Additionally, WO 2005/116064 is related to the covalent fibronectin binding characteristic with change rather than only fibrin
The mutant of former binding characteristic.Additionally, the document proves whether mutain and the combination of part are reduced not over experiment, and
And without any supportive immunogenicity data of offer.
Therefore, in view of multi-drug resistant in gram-positive bacteria prevalence and to these multi-drug resistant bacteriums into
Work(treat and vaccine shortage, it is any to use antibiotic and will have processing the surrogate therapeutic of such bacterium infection
Significance.
Additionally, will be significant for any for the treatment of or any improvement of the effect of vaccine, particularly exist
In clinical setting.
Therefore, the present invention is intended to provide the substituting and improved cure of the such bacterium infection for the treatment of.
The content of the invention
First of the invention general aspect, there is provided the restructuring grape ball that the combination to its host's part is reduced
Bacterium MSCRAMM or MSCRAMM sample albumen or its fragment, for treating.
According to preferred embodiment, there is provided the restructuring staphylococcus fiber of the ability without binding fiber proteinogen
Proteinogen associativity MSCRAMM albumen or its fragment for including at least a portion fibrinogen calmodulin binding domain CaM, for treating.
According to the second aspect of the invention, there is provided the method and/or treatment that immune response is induced in individuality suffer from micro-
The method of the patient of biological infection, it includes giving the restructuring staphylococcus that the individual combination to its host's part is reduced
MSCRAMM or MSCRAMM samples albumen or its fragment, or comprising it is described restructuring staphylococcus MSCRAMM or MSCRAMM sample albumen or
The vaccine of its fragment.
According to the third aspect of the invention we, there is provided comprising the restructuring staphylococcus that the combination to its host's part is reduced
The vaccine of MSCRAMM albumen or its fragment.
According to the fourth aspect of the invention, there is provided for the restructuring staphylococcus that the combination to its host's part is reduced
MSCRAMM or MSCRAMM samples albumen or its fragment and the antibody that produces, the preferably form of hyper-immuneserum.
According to the fifth aspect of the invention, there is provided Immunogenic agents composition, it includes the knot to its host's part
Close restructuring staphylococcus MSCRAMM or MSCRAMM the sample albumen or its fragment and pharmaceutically acceptable adjuvant for reducing.
Describe in detail
In this manual, will be understood as can phase for term " adhesin ", " MSCRAMM " and " Cell wall anchored proteins "
Mutually replace and cover all of ligand binding protein from microorganism.It is desirable that these albumen and fibrinogen, blood red
Element or hemoglobin, Hapto-globin-haemoglobin, hemin, collagen and other such ligand bindings.Term
" MSCRAMM samples " albumen is intended to such MSCRAMM albumen there is related amino acid sequence, similar module to design
And/or the albumen or adhesin of common/similar binding structural domain group structure (organization), such as Isd albumen.It is preferable
There is ground, MSCRAMM sample albumen similar binding structural domain group structure/module to design.In addition, MSCRAMM samples albumen and MSCRAMM
Albumen can have at least 50%, preferably 60%, preferably 75%, more preferably 85%, even more preferably from 95%, and more preferably 99% or
Amino acid sequence identity higher.
It should also be understood that any percentage identity or homology alleged by this specification are to use obtainable routine side
What method determined on the whole/complete length of sequence.
Term " microorganism (micro-organism) ", " microorganism (microbe) ", " (microbial) of microorganism "
Or similar term includes but is not limited to following organism, including：Bacterium, fungi, virus, yeast and/or mould.
Term " immune effective quantity " includes stimulating the those amounts of B cell and/or t cell responses.
First of the invention general aspect, there is provided the restructuring grape ball that the combination to its host's part is reduced
Bacterium MSCRAMM or MSCRAMM sample albumen, or its fragment, it includes at least a portion ligand binding region, for treating.So
Recombinant protein can be used to treat microorganism infection, for example treat pyemia, septic arthritis and/or endocarditis or other
Similar symptom or morbid state.Such microorganism infection is desirably caused by staphylococcus or other similar microorganisms
's.
A particular implementation according to this aspect of the invention, the restructuring MSCRAMM or MSCRAMM sample albumen
Or its fragment has reducing or does not possess ability with its host's part Non-covalent binding.
It should therefore be understood that restructuring staphylococcus MSCRAMM or MSCRAMM sample albumen or its fragment may have reducing
And its host's part combination or be prevented from the combination of its host's part.
According to inference of the present invention：MSCRAMM or MSCRAMM samples albumen is by depressed place, lock and locking (Dock, Lock and
Latching (DLL)) may be reduced or prevent with the Non-covalent binding of generation in its ligand binding processes.It is verified
MSCRAMM is related to the noncovalent interaction carried out by DLL models with first step of its ligand binding.These are
The primary noncovalent interaction of MSCRAMM and part.The final stage of MSCRAMM- ligand bindings is related to covalent phase interaction
With.In this particular implementation, due to change depressed place, lock and locking, restructuring MSCRAMM or MSCRAMM samples albumen or its
Fragment has reducing or does not possess ability with its host's part Non-covalent binding.One or more depressed places, lock or locking step
Can be changed.
DLL models be the compound from SdrG Yu its part three-dimensional structure in illustrate.It has now been shown that ClfA is logical
The minor variations for crossing DLL mechanism play a role (Ganech et al. (2008) " Astructural model of the
Staphylococcus aureus Clfa-fibrinogen interaction opens new avenues for the
design of anti-staphylococcal therapeutics”.PloS Pathog4(11)；e1000226).DLL moulds
Type more particularly to participates in the noncovalent interaction of ligand binding.For all other similar structure type (either according to
Amino acid similarity/homology is also according to structure group structure homology) albumen infer with DLL models, including but do not limit
In MSCRAMM or MSCRAMM sample albumen.
Especially when MSCRAMM ClfA/ClfB are related to, the subprovince domain of minimum ligand binding domains inclusion region A is found
N1 to N3, the subprovince domain N2 and N3 for particularly being folded comprising modification D ev-IgG Ig.It is immune ball that modification D ev-IgG Ig are folded
The neomorph of protein motif, it is also referred to as DE variants.According to inference, formed between two DEv-IgG domains of ClfA/B
Hydrophobic pocket be fibrinogen γ-chain ligand binding site.Substantially, part is combined with the drain tank for separating N2 and N3.
Especially, in ligand binding processes, the unfolded peptide components insertion of part is located at the groove between N2 and N3 subdomains.Asia knot
The locking peptide occurred conformation of the C-terminal of structure domain N3 changes and inserts between two B chains of subdomain N2, therefore, part is locked
It is scheduled on appropriate location.Really, in clumping factor residue Tyr256, Pro336, Tyr338 and Lys389 (they be considered as with
The γ chain end residues of fibrinogen408AGDV411Contact) mutation replace cause the albumen lose or significantly reduce it to fibre
The former affinity of fibrillarin.The more details of the specific embodiment are discussed further below.
Although these teachings are related to clumping factor, especially ClfA, they are equally applicable to other has similar mould
Block binding structural domain group structure and in a similar manner with MSCRAMM the and/or MSCRAMM sample albumen of ligand binding.
Therefore, in order to provide restructuring staphylococcus MSCRAMM or MSCRAMM the sample egg to the combination reduction of its host's part
White or its fragment, thus it is possible to vary full-length proteins, ligand binding domains, minimum ligand binding domains or its fragment reducing or
Prevention and the combination of its host's part.It is desirable that for ClfA/ClfB or other similar MSCRAMM or MSCRAMM samples albumen,
The subprovince domain N2 and N3 (it is ideally folded comprising modification D ev-IgG Ig) of region A can be changed to prevent or reduce and its place
The combination of main part.Such change is designed to prevent the combination of part and drain tank, and the drain tank separates needed for DLL
Minimum ligand binding domains.
Can be using full-length proteins, ligand binding domains, minimum ligand binding domains or its fragment, by amino acid
Substitution is deleted, and makes ligand binding domains that such change occur in amino acid levels.It should be understood that can also use and match somebody with somebody
Binding protein precursor has the albumen or its fragment of sufficiently high homology.High homology defined herein refers to, in full length DNA sequence
At least 50% on row, preferably 60%, preferably 70%, preferably 80%, more preferably 90%, even more preferably from 95% and more preferably 95%
To 99% and the matching of more preferably 99% or more nucleotides, or, refer to when amino acid sequence, amino acid sequence
Though row are differed but produce the albumen with same functionality and activity.It should be understood that these discussions on high homology
Suitable for the three-dimensional structure of protein, i.e. module binding structural domain group structure.
It should be appreciated that, it is possible to use complete ligand binding protein, ligand binding domains, minimum ligand binding domains
Or its fragment.
Using the albumen such as ligand binding domains of the truncation of ligand binding protein, minimum ligand binding domains or make
With its fragment is for ease of handling and to overcome the other problems such as undesired Protein cleavage be favourable.For example, depositing
It is that locking peptide in minimum ligand binding domains can be deleted/remove or change.For example, locking peptide correspondence in ClfA
In the amino acid 532 to 538 of region A；Corresponding to (Walsh et al. (2004) of amino acid 530 to 540 of region A in ClfB
JBC 279(49)：50691-50699).These residues can be changed, replaced or removed/deleted to prevent part from passing through DLL
Combined with MSCRAMM.In this manner it is achieved that MSCRAMM is prevented from the DLL " locking " of its part.This " locking " is by non-
The mode of covalent interaction occurs.In one embodiment, will be all along the C-terminal amino acid residue of remaining area A
Locking peptide is removed.According to another embodiment, only locking peptide region is removed.According to another implementation method, locking
There is 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor to reduce or prevent ligand binding/locking in peptide region.These discuss be applied to it is all of by DLL or class
Like model and MSCRAMM the or MSCRAMM sample albumen of ligand binding.
By changing MSCRAMM or MSCRAMM sample albumen by this way, can provide and not have and its ligand binding energy
The ligand binding protein of power, it can stimulate the bigger immune response of producing ratio wild-type protein after immune.Advantageously, this subtracts
Systemic inflammatory is lacked, so as to reduce microorganism virulence.Therefore, this part lacked with the change of its ligand binding capacity
Associativity MSCRAMM or MSCRAMM sample albumen is advantageously used for the treatment of microorganism infection.Therefore, these discoveries are presented
The new and valuable vaccine/immunotherapeutic agent of bacterium infection is resisted, with the vaccine from wild-type protein or immunization therapy
Agent is compared, and it provides more preferable effect.
An implementation method of the invention, the part is ferroheme, hemoglobin or fibrinogen.Can examine
Consider other parts, such as Hapto-globin-haemoglobin, hemoglobin, hemin, collagen etc..
According to another implementation of the invention, restructuring MSCRAMM albumen is selected from：Fibrinogen binding protein；Or
SdrD, SdrE, SdrG and/or SdrF.
Detailed fibrinogen binding protein above, including but not limited to ClfA, ClfB, FnBPA, FnBPB,
FbI, IsdA etc..Have shown that SdrG/F is combined with collagen.Other MSCRAMM include SasA, SasG, SasK and SdrH.
Restructuring MSCRAMM samples albumen can be selected from：IsdA, IsdB and/or IsdH.
Based on the discovery from fibrinogen associativity MSCRAMM ClfA, can for example include IsdH's and IsdB
Similar non-ligand-binding mutant is produced in NEAT (NEAr transhipments) motif of Isd albumen.As set forth in detail above, IsdA
With IsdB without the structure with Clf or Sdr albumen same types.But, the NEAT motifs in Isd directly participate in ligand binding
(Hapto-globin-haemoglobin, hemoglobin, hemin), therefore, change in NEAT motifs will be changing Clf or Sdr
The same way that interacts of DLL or DLL sample hosts part prevent host part from interacting.Much contain NEAT domains
Albumen participate in the combination of ferroheme (haem), including the IsdA in staphylococcus aureus.According to inference：IsdA and ferroheme knot
The property of conjunction is contained in NEAT domains.Apo-IsdA NEAT domains and with the compound of ferroheme in crystal structure
Have revealed that out the clathrin adaptation increment sandwich foldings of β with big hydrophobicity ferroheme binding pocket.IsdB has two
NEAT motifs；IsdA has a NEAT motif.The residue of ligand binding can be predicted as by changing to separate Isd albumen
Non- ligand-binding mutant, for example, carried out by changing the residue between β chains and/or hydrophobic pocket.Furthermore it is possible to change
NEAT motifs interact influenceing non-covalent host-part.
Another of this aspect of the invention immune response and/or is controlled embodiment there is provided being induced in individuality
The method for treating the patient with microorganism infection, it includes giving the restructuring Portugal that the individual combination to its host's part is reduced
Grape coccus MSCRAMM or MSCRAMM sample albumen or its fragment, or the restructuring grape ball reduced comprising the combination to its host's part
The vaccine of bacterium MSCRAMM or MSCRAMM sample albumen or its fragment.
Another of this aspect of the invention is embodiment there is provided comprising the combination reduction to its host's part
The vaccine of restructuring staphylococcus MSCRAMM or MSCRAMM sample albumen or its fragment.
Another of this aspect of the invention is embodiment there is provided for the combination reduction to its host's part
The antibody for recombinating staphylococcus MSCRAMM or MSCRAMM sample albumen or its fragment and producing, the preferably shape of hyper-immuneserum
Formula.
Embodiment there is provided Immunogenic agents composition, it includes right another of this aspect of the invention
Restructuring staphylococcus MSCRAMM or MSCRAMM sample albumen or its fragment that the combination of its host's part is reduced.
Preferred embodiment of the invention, there is provided the restructuring grape without the ability combined with fibrinogen
Coccus fibrinogen binding protein, or it includes the fragment of at least a portion fibrinogen calmodulin binding domain CaM, for treating.
It should be understood that restructuring staphylococcus fibrinogen binding protein or its fragment can be used to treat microorganism infection,
Preferably staphy lococcus infection, for example treat pyemia, septic arthritis and/or endocarditis or other similar symptom or
Morbid state.
The fibrinogen calmodulin binding domain CaM of albumen is changed so as to no longer be combined with fibrinogen.As described above, this changes
Change can occur in nucleotides or amino acid levels.It should be understood that can also use that there is foot with fibrinogen binding protein
The albumen or its fragment of enough homologys high.High homology defined herein refers to, at least 50% in full length DNA sequence, excellent
Select 60%, preferably 70%, preferably 80%, more preferably 90%, even more preferably from 95%, not only more preferably 95% to 99% but also more preferably
The matching of 99% nucleotides, or, refer to when being used on amino acid sequence, though amino acid sequence differs generation tool
There is the albumen of same functionality and activity.It should be understood that these discussions on high homology relate to the three of protein
Dimension structure.
It should be appreciated that, it is possible to use complete fibrinogen binding protein, fibrinogen calmodulin binding domain CaM, minimum fiber
Proteinogen calmodulin binding domain CaM or its fragment.Such as it is not intended to for ease of handling and overcoming using the albumen or its fragment that truncate
The other problems such as Protein cleavage be favourable.This will be explained below.
It is desirable that such fragment should include at least a portion fibrinogen calmodulin binding domain CaM of MSCRAMM.Using cut
Short albumen or its fragment (comprising one or more subdomains for for example only having part-fibrinogen calmodulin binding domain CaM) it is good
Place is that it is possible in the case where not degrading with high yield purifying protein.The fibrinogen calmodulin binding domain CaM of ClfA albumen,
Or referred to as a-quadrant, comprising 3 subprovinces domain：N1, N2 and N3.Therefore, immunogenic fragments can include the Asia of ClfA a-quadrants
Region N1, N2 and/or N3 or its fragment.Thus, for example, when being related to ClfA, fragment can be with inclusion region A or many
Individual subdomain, N1, N2 or N3.Ideally, it is possible to use N2 and N3, because the truncate less easily occurs proteolysis
(it has been reported that there is proteolytic cleavage site between N1 and N2 in ClfA and ClfB) and can in Escherichia coli with
Higher level is expressed.N2 and N3 are the minimum fibrinogen calmodulin binding domain CaMs of Clf albumen.
It should be appreciated that, although following discussion is related to fibrinogen binding protein ClfA, but these discussions are equally applicable
It is particularly other to exist with ClfA on amino acid or protein structure level in other MSCRAMM, MSCRAMM sample albumen
Similar fibrinogen binding protein in structure, such as ClfB, FbI and SdrF/G (it is also combined with collagen).Additionally, these
Teaching is applied to FnBPA and FnBPB.Therefore, although discussion below is related to fibrinogen binding protein, but they are similarly
Suitable for MSCRAMM the or MSCRAMM sample albumen of the ligand binding outside other and fibrinogen.
It has been unexpectedly discovered that：After immune, it is this without with the change of fibrinogen binding ability
Fibrinogen binding protein, truncate or its fragment stimulate producing ratio, and those are bound to the open country of fibrinogen in the normal fashion
The bigger immune response of raw type albumen.Advantageously, when being expressed by staphylococcus aureus, the fibrinogen knot of this change
Hop protein does not cause systemic inflammatory.Therefore, microorganism virulence reduction.Therefore, this shortage and fibrinogen binding ability
The albumen of change can be advantageously used in treatment microorganism infection.It is contrary to expectations, it has been found that, the fibrin of change
Former protein-bonded protective effect is bigger than wild-type protein.We have found that the drug regimen of the recombinant protein comprising such change
The medicine of thing or vaccine ratio comprising the identical recombinant protein (such as ClfA, ClfB, SdrG etc.) of the form of (wild type) without alteration
Compositions or vaccine are more efficient.
Therefore, these discoveries present the new and valuable vaccine/immunotherapeutic agent of resistance bacterium infection, and same
Wild-type protein as vaccine/immunotherapeutic agent is compared, and it provides more preferable effect.
It should be understood that the albumen for changing, either MSCRAMM or MSCRAMM samples or fibrinogen or other part knots
Close, can be used to produce antibody, including monoclonal, polyclonal, chimeric, humanized antibody or its fragment, it is such for treatment
Microorganism infection.Then the composition including such antibody such as hyper-immuneserum can be provided, these compositions can be used for
The patient of staphy lococcus infection has been infected in treatment.
Therefore, albumen or its active fragment can be used for the combination of suppression staphylococcus and extracellular matrix (ECM) and be used for
Staphy lococcus infection in preventing/treating patient.
Additionally, albumen or its active fragment and the antibody for the albumen can be used to treat from staphy lococcus infection
Infection, carry out the vaccine of actively or passively vaccine inoculation for developing, when as pharmaceutical composition to wound or medicine equipment
Using when, albumen and antibody can be employed as preventing the blocking agent of microorganism infection.For example, these albumen or its fragment can be used to lead
Dynamic vaccine, the antibody for these albumen can be used for passive vaccine.
These vaccines described herein and product show significantly improving for prior art, teaching in prior art
MSCRAMM carries out immune general purposes, but does not instruct described herein unexpected and improved vaccine or product
Product.
What the preparation of protein, DNA and antibody was well-known in the art, will not be described in detail again herein.Producing this
Routine techniques is ideally used during a little molecules.It should also be appreciated that the present invention covers comprising target nucleic acid or amino acid sequence
Nucleic acid construct, comprising such nucleic acid construct expressing the recombinant host cell and IMMUNOGENIC COMPOSITION of target protein
Thing.
In order to be administered, protein composition can be scattered in aseptic isotonic salting liquid or other pharmaceutically acceptable assistants
In agent.
It should be understood that vaccine can be DNA or protein vaccine.
Immunity inoculation can be carried out by injecting DNA, protein or antibody.It is alternatively possible to give including and express DNA
Attenuation work organism.
The amount of DNA, protein or the antibody that can give will depending on several mitigation key elements, including, it is strong for promoter
The dependence of the immunogenicity of the gene of degree, protein expression and expression.For each new application can change these factors with
Immune effective quantity the need for being wanted.
According to another implementation of the invention, there is provided immune response and/or treatment are induced in individuality with micro-
The method of the patient of biological infection, it includes giving the individuality and does not possess restructuring grape ball with fibrinogen binding ability
Bacterium fibrinogen binding protein or its fragment for comprising at least fibrinogen calmodulin binding domain CaM.
A kind of another preferred embodiment of the invention, there is provided vaccine, it is included not possesses and fibrin
The restructuring staphylococcus fibrinogen binding protein of former binding ability or its contain at least a portion fibrinogen land
The fragment in domain.
Preferred embodiment according to another preferred, there is provided combined for restructuring staphylococcus fibrinogen
The antibody that albumen or its fragment for containing at least a portion fibrinogen calmodulin binding domain CaM are produced, the preferably shape of hyper-immuneserum
Formula, the restructuring staphylococcus fibrinogen binding protein or its fragment do not possess the ability combined with fibrinogen.
Preferred embodiment according to another preferred, there is provided a kind of Immunogenic agents composition, it is included
Do not possess and contain at least a portion with the restructuring staphylococcus fibrinogen binding protein of fibrinogen binding ability or its
The fragment of fibrinogen calmodulin binding domain CaM and pharmaceutically acceptable adjuvant.
It is desirable that restructuring staphylococcus fibrinogen binding protein or its fragment are derived from staphylococcus aureus, epidermis
Staphylococcus and/or road Deng staphylococcus.
The fibrinogen binding protein of these implementation methods can selected from following FbI, SdrF and/or SdrG (its also with
Collagen combination) one kind.Alternatively, fibrinogen binding protein can be selected from following one kind：Fibrinogen binding protein
Clumping factor A (ClfA), fibrinogen binding protein clumping factor B (ClfB), fibronectin-fibrinogen binding protein
A (FnBPA), fibronectin-fibrinogen binding protein B (FnBPB).IsdA promotes adhesion, and for fibrinogen
Compatibility with fibronectin is weaker, so can technically be defined as fibrinogen associativity MSCRAMM.
It should be understood that nucleotides or amino in the fibrinogen calmodulin binding domain CaM of such fibrinogen binding protein
The recombinant protein that will be produced and do not possess with fibrinogen binding ability is deleted in acid substitution.
The combination for having reasoned out ClfA- fibrinogens is by the depressed place similar to SdrG, lock and locking (DLL) mechanism
Occur.DLL models as detailed above.The region A of ClfA is responsible for protein ligand interaction.As shown in Figure 11, several fibres
The modular structure of fibrillarin original associativity MSCRAMM is similar, all contains the region A similar to ClfA.
Fibrinogen γ-chain binding site peptide point is located in the drain tank of the node between the N2 and N3 of ClfA.Therefore, with
On the substitution that refers to or deletion be designed to change MSCRAMM protein ligands and interact and prevent ClfA and fibrinogen
Non-covalent binding.
A specific embodiment of the invention, restructuring staphylococcus fibrinogen binding protein is the fibre of ClfA
Fibrillarin original binding deficient type mutant.In this embodiment, the fibrinogen calmodulin binding domain CaM A of ClfA is changed by any mode
Become (for example replace or delete mutation) so as to it is no longer combined with fibrinogen.
It is desirable that fibrinogen binding protein is ClfA, but, ClfA has and is combined with many other fibrinogens
The similar three-dimensional structure of albumen.It should therefore be understood that these discussions on ClfA are equally applicable to other MSCRAMM fibers
Proteinogen associated proteins, including ClfB, FnBPA, FnBPB, FbI, SdrG/F, IsdA etc..All these albumen all have similar
Three-dimensional structure, therefore, it can carry out fibrinogen calmodulin binding domain CaM similar change/mutation reach identical result.
ClfA is 993 albumen of amino acid, and it includes 520 fibrinogen binding structural domains of amino acid (from ammonia
Base acid 40 to 559).The fibrinogen binding structural domain is the N-terminal A domains comprising subprovince domain N1, N2 and N3.Across N1
The whole fibrinogen region from amino acid 40 to amino acid 559 to N3 can be used for the present invention.It is alternatively possible to use N1
To the truncate in N3 regions, such as 221 to 559 (minimum fibrinogen calmodulin binding domain CaMs), 221 to 531 (without locking peptide and its
The minimum fibrinogen region of residue afterwards) etc..Ideally, it is possible to use subprovince domain N2 and N3, minimum fibrinogen knot
Region is closed, it corresponds to amino acid residue 221 to 559.It is alternatively possible to use the fragment in these subprovince domains.
Research has shown that, the amino acid residue 221 to 559 for covering N2 and N3 regions of ClfA with fibrinogen
Played a significant role with reference to during, it is minimum fibrinogen calmodulin binding domain CaM.We have also unexpectedly found that the area
But amino acid residue mutation in domain produces the expression egg that can be recognized by host immune defenses be combined without fibrinogen
In vain, therefore, reduce correlation virulence.This region (339 fibrinogen binding structural domains of amino acid) tool of ClfA
There is specific three-dimensional structure, i.e., so-called DE variants IgG is folded, and it is minimum Fg combinations truncate, if being changed
(by substitution or deletion etc.), using the teaching of the invention it is possible to provide improved treatment.
Can be changed to lead by nucleotides or amino acid levels being replaced, being added or being inserted or deleted
Cause the forfeiture of fibrinogen binding activity.It is desirable that replacing to the three-dimensional structure of albumen or fragment (for example, so-called DE becomes
Body IgG is folded) adversely affect, so it can not be combined with fibrinogen again.
It is desirable that nucleotides or 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor reduce the noncovalent interaction with fibrinogen, preferably by resistance
Only the hydrophobic pocket of the N2 and N3 of part region A protein-bonded with septate fibre proteinogen is combined.Alternatively, corresponding to amino acid
532 to 538 locking peptide region can be changed by substitution or deletion, to prevent ligand binding.Further, it is possible to use not having
Locking peptide region and the remainder optionally without C-terminal protein residues are the truncation without amino acid residue 532 to 559
Thing/fragment.
A specific embodiment according to this aspect of the invention, can be by respectively by amino acid P336Replace with
Serine and/or by amino acid Y338Aspartic acid is replaced with to build the mutant of the fibrinogen binding deficient of ClfA.It is residual
The selection of base is x-ray crystal structure and " to the single change reduction binding activity of proline or tyrosine " based on ClfA
Observation.It is surprising that we have found that mutant ClfA albumen (rClfAP336S Y338A) immune response stimulating and
Can be used to produce significantly more efficient vaccine or Antybody therapy.The substitution can occur total length fibrinogen binding protein,
In fibrinogen calmodulin binding domain CaM, minimum fibrinogen calmodulin binding domain CaM or its fragment.
Another embodiment according to this aspect of the invention, can be by respectively by amino acid P336Replace
For aspartic acid and/or by amino acid Y338Serine is replaced with to build the mutant of the fibrinogen binding deficient of ClfA.
As an ibid implementation method, mutant ClfA albumen (rClfAP336A Y338S) can also be used for generation significantly more efficient
Vaccine or Antybody therapy.
Or, the change can be the form deleted, including the fibre without locking peptide sequence (amino acid 532 to 538)
Fibrillarin original calmodulin binding domain CaM, to produce the recombinant fibrinogen combination egg not possessed with fibrinogen Non-covalent binding ability
In vain.In this embodiment, the amino acid residue 221 to 531 of the region A of ClfA has been used, it does not have locking peptide and C thereafter
Terminal residue.It is alternatively possible to consider the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in locking peptide ammino acid 532 to 538, it prevents fibrinogen
DLL。
It should be understood that all albumen in Clf-Sdr families all pass through DLL models and ligand binding.It is three-dimensional by simulating
Structure can predict locking peptide and prepare the truncate without locking peptide, whether total length (N1 to N3) or minimum ligand binding
Truncate N2-N3 or its fragment.
We have found that the rClfA albumen (whether deleting mutant, substitution or truncate) of these substitutions reduces virulence
With disease consequence, and the astoundingly few systemic inflammatory of induction ratio wild-type protein.
Therefore, based on the albumen surveyed, it is contemplated that recognize mutation simultaneously with immune will the enhancing that these mutant proteins are carried out
The antibody level of body and wild-type protein and the immune response bigger than wild-type protein will be provided.
Therefore, by changing so as to its ClfA for no longer being combined with fibrinogen is for having for being actively or passively immunized
Candidate therapeutic agent.In this manner it is achieved that the ClfA albumen through changing can be used as vaccine in itself, or can be using for this
Plant the antibody that the ClfA albumen for changing is produced.As described above, vaccine can be DNA or protein vaccine.
The following sequence listed in following table can be with used according to the invention.
1(N- ends extend (6 × His labels and other residue) and include 6 His residues other N residues, are followed by
Gly and Ser.Other C-terminal residue includes Lys, and being followed by Leu (can be using other other N and C-terminal residue-take
Certainly in the N/C end tags of primer or needs used)
2Other N residues (6 × His labels and other residue) include 6 His residues, are followed by Gly and Ser.Separately
Outer C-terminal residue includes Arg, and being followed by Ser (can use other other N and C-terminal residue-depend on used
The N/C end tags of primer or needs)
3Without the locking peptide corresponding to amino acid residue 532 to 538 and remaining a-quadrant C-terminal residue, i.e., no ammonia
Base acid residue 532 to 559.
It is desirable that restructuring staphylococcus fibrinogen binding protein is included according to any SEQ ID NO：1 to 3 amino
Acid sequence or its fragment, wherein residue P336And/or Y338Replaced by serine and/or alanine.
Alternatively, the fragment of restructuring staphylococcus fibrinogen binding protein is included according to any SEQID NO：4 to
SEQ ID NO：14 amino acid sequence.SEQ ID NO：4 and 5 correspond only to ClfA domain N1, N2, N3, and it is respectively
rClfA P336S Y338A and rClfA P336A Y338S, as shown in upper table.
Speculated based on the substitution in the locking carried out in SdrG：The existing defects in terms of conformation change or β chain complementations
Being substituted in ligand binding in locking also will be defective.Therefore, it is desirable that substitution betides the ammonia corresponding to locking peptide
Base acid residue 532 to 538 and influence peptide occurred conformation change ability or and part combination or the two all have.Alternatively, change
Become can be comprising amino acid residue 532 to 538 (Δ locking peptide) be removed, to produce similar result together.In addition, without amino
The C-terminal truncated mutant of sour residue 532 to 559 (including locking peptide residue) also will influence and the combination of part.
It should, however, be considered that arrive, in addition to those specialized more than, it is also possible to which other amino acid residues are entered
Row substitution.For example, can be replaced to change albumen or its fragment to Glu 526, Val 527, Tyr 256 and Lys 389
Fibrinogen binding property.Therefore, any substitution for reducing binding ability can be taken into account.It is desirable that such
Substitution or delete influence hydrophobic pocket and it is associated with hydrophobic ditch in ligand binding mechanism, such as the homologue in ClfA
N526 in Val527 and ClfB.In ClfB, research has shown that Q235 and N526 reduces combination.Phase has been carried out with FnBPA
Like studying, wherein it is important to show that N304 and F306 are combined for Fg.Therefore, the mutation in these amino acid residues will influence
Ligand binding.
It should be understood that these are discussed for other fibrinogen binding proteins such as ClfB, SdrG, FnBPA, FnBPB
It is equally applicable.Therefore, treatment (vaccine, antibody or pharmaceutical composition etc.) can include complete fibrinogen calmodulin binding domain CaM or
Its fragment.
In this manual, term " including (comprise), including (comprises), including (comprised) and bag
Include (comprising) " or its any variant and term " including (include), including (includes), include
(included) and including (including) " or its any variant be considered as that can be used interchangeably completely, they should
It is endowed most probable explanation wide.
The invention is not restricted to embodiment described above, can within the scope of the claims in terms of explanation and details
Change.
The present invention will be described by reference to following non-limitative drawings and embodiment now.
Fig. 1 to 15 shows the result of embodiment 1.
Fig. 1 shown being vaccinated with staphylococcus aureus strains Newman, and clfAPYI, clfAPYII and clfA without
Imitate the arthritic seriousness (A) and weight loss (B) determined with arthritis index in the mouse of mutant.Inoculation 3.2 ×
106-6.0×106The staphylococcus aureus strains of cfu.Data are expressed as between median (square or center line), quartile
Away from (frame) and 80% middle ware away from (palpus).Collect from three data of experiment.NNewman=27-30, NclfAPYI=30,
NclfAPYII=10, NclfA=16-20.
Fig. 2 is shown with 3.2 × 106-6.0×106The staphylococcus aureus strains Newman of cfu, and clfAPYI,
Bacterial growth after the inoculation of clfAPYII and clfA null mutants in the mouse kidney of 7-8 days.Data are expressed as each pair kidney
Cfu in dirty.When can't detect growth, the highest possible counting for being set to the dilution factor according to used by will be counted.Collect and
From three data of experiment.NNewman=26, NclfAPYI=30, NclfAPYII=10, NclfA=15.
Fig. 3 shows and is inoculated with 5.2,5.1 or 3.3 × 10 respectively7The staphylococcus aureus strains Newman of cfu,
Mouse survival situation after clfAPYI mutant or clfA null mutants.Every group of N=10 from the outset.
Fig. 4 shows and is inoculated with 9.4,7.9,10.7 or 9.8 × 10 respectively6The staphylococcus aureus strains LS-1 of cfu, and
Mouse survival situation after clfAPYI, clfAPYII or clfA null mutant.Every group of N=15 from the outset.
Fig. 5 shows immune with BSA, recombinant C lfA or recombinant C lfAPY (i.e. ClfAPYI recombinant protein As domain) and connects
Plant 2.3 × 107The Survival of the mouse of the staphylococcus aureus Newman of cfu.Every group of N=15 from the outset.
Fig. 6 shows and is vaccinated with 3.2 × 106-6.0×106The staphylococcus aureus strains Newman wild types of cfu, and
The frequency of the arthritic mice of clfAPYI, clfAPYII and clfA null mutant.Collect from three data of experiment.
NNewman=27-30, NclfAPYI=30, NclfAPYII=10, NclfA=16-20.
Fig. 7 shows and be vaccinated with respectively 5.2,5.1 or 3.3 × 107The staphylococcus aureus strains Newman of cfu is wild
The arthritic seriousness determined with arthritis index in the mouse of type, clfAPYI mutant or clfA null mutants.Data
Median (square), interquartile range (frame) and 80% middle ware are expressed as away from (palpus).NNewman=0-10, NclfAPYI=
9-10, NclfA=0-10.
Fig. 8 shows and be vaccinated with respectively 5.2,5.1 or 3.3 × 107The staphylococcus aureus strains Newman of cfu is wild
Weight loss in the mouse of type, clfAPYI mutant or clfA null mutants.Data are expressed as median (center line), four
Quantile spacing (frame) and 80% middle ware are away from (palpus).NNewman=0-10, NclfAPYI=9-10, NclfA=0-10.
Fig. 9 shows immune with BSA, recombinant C lfA or recombinant C lfAPY (i.e. ClfAPYI recombinant protein As domain) and connects
Plant 4.0 × 106With the arthritic serious of arthritis index measure in the mouse of the staphylococcus aureus Newman of cfu
Property.Data are expressed as median (square), interquartile range (frame) and 80% middle ware away from (palpus).From the outset, every group
NBSA=14, NclfAPY=14, NclfA=15.
Figure 10 gives wild type ClfA A domain proteins (rClfA), the only nucleotides and amino acid of domain N123
Sequence, wherein having marked the residue for being altered to create rClfAPYI/II (SEQ ID No.3) in the examples below with highlighted
(P336And Y338).It is used for exactly this recombinant protein A domain of vaccine inoculation in the examples below.
Figure 11 shows the exemplary diagram of FnBPA, ClfB, ClfA and SdrG protein structure.Region A is fibrinogen knot
Region is closed, S is signal sequence, and W is cell membrane Oil pipeline domain, and M is film grappling, and it includes LPXTG motifs ,+represent positively charged
Residue, R is repeat region.In ClfA, region A includes N123 (not shown)s.BCD regions (and the FnBPB of FnBPA
Shorter CD regions-do not show) combined with fibronectin.
Figure 12 is shown in second booster immunization 9 days afterwards, for bovine serum albumin(BSA) (BSA), recombinant C lfA40-
Recombinant C lfAPY40-559's in 559 (rClfA) or the immune mice serum samples of recombinant C lfAPY40-559 (rClfAPY)
Specific antibody reacts, and 1 day afterwards with 2.3 × 107Cfu/ mouse carry out staphylococcus aureus strains Newman wild types
Infect to induce pyemia.Data are expressed as median (center line), interquartile range (frame) and 80% middle ware away from (palpus).
NBSA=13-15, NrClfA=15, NrClfAPY=15.
Figure 13 is shown in second booster immunization 9 days afterwards, for bovine serum albumin(BSA) (BSA), recombinant C lfA40-
The spy of the recombinant C lfA40-559 in 559 (rClfA) or the immune mice serum samples of recombinant C lfAPY40-559 (rClfAPY)
Heterogenetic antibody reacts, and 1 day afterwards with 2.3 × 107Cfu/ mouse carry out the sense of staphylococcus aureus strains Newman wild types
Contaminate to induce pyemia.Data are expressed as median (center line), interquartile range (frame) and 80% middle ware away from (palpus).NBSA
=13-15, NrClfA=15, NrClfAPY=15.
Figure 14 is shown in second booster immunization 9 days afterwards, for bovine serum albumin(BSA) (BSA), recombinant C lfA40-
Recombinant C lfAPY40-559's in 559 (rClfA) or the immune mice serum samples of recombinant C lfAPY40-559 (rClfAPY)
Specific antibody reacts, and 1 day afterwards with 4.0 × 106Cfu/ mouse carry out staphylococcus aureus strains Newman wild types
Infection is with triggering septic arthritis.Data be expressed as median (center line), interquartile range (frame) and 80% middle ware away from
(palpus).NBSA=14-15, NrClfA=15, NrClfAPY=15.
Figure 15 is shown in second booster immunization 9 days afterwards, for bovine serum albumin(BSA) (BSA), recombinant C lfA40-
The spy of the recombinant C lfA40-559 in 559 (rClfA) or the immune mice serum samples of recombinant C lfAPY40-559 (rClfAPY)
Heterogenetic antibody reacts, and 1 day afterwards with 4.0 × 106Cfu/ mouse carry out the sense of staphylococcus aureus strains Newman wild types
Dye is with triggering septic arthritis.Data be expressed as median (center line), interquartile range (frame) and 80% middle ware away from
(palpus).NBSA=14-15, NrClfA=15, NrClfAPY=15.
Figure 16 of embodiment 2 is shown in second booster immunization 9 days afterwards, for bovine serum albumin(BSA) (BSA), weight
Group ClfA221-559 (rClfA221-559) or the immune mice serums of recombinant C lfAPY221-559 (rClfAPY221-559)
The specific antibody reaction of the recombinant C lfAPY221-559 in sample.Data are expressed as median (center line), interquartile range
(frame) and 80% middle ware are away from (palpus).NBSA=15, NrClfA221-559=14-15, NrClfAPY221-559=14-15.
Figure 17 of embodiment 2 is shown in second booster immunization 9 days afterwards, for bovine serum albumin(BSA) (BSA), weight
Group ClfA221-559 (rClfA221-559) or the immune mice serums of recombinant C lfAPY221-559 (rClfAPY221-559)
The specific antibody reaction of the recombinant C lfA221-559 in sample.Data are expressed as median (center line), interquartile range
(frame) and 80% middle ware are away from (palpus).NBSA=15, NrClfA221-559=14-15, NrClfAPY221-559=14-15.
Figure 18 of embodiment 3 is shown in second booster immunization 9 days afterwards, for using recombinant C lfAPY221-531
(rClfA221-531) the specific antibody reaction of the recombinant C lfA221-53l in immune mice serum sample.Data are represented
It is median (center line), interquartile range (frame) and 80% middle ware away from (palpus).NrClfA221-531=14-15.
Embodiment
Embodiment 1
RClfA a-quadrants truncate comprising N1, N2 and N3 (amino acid 40-559)
Material and method
The full details of the digital reference document in providing the bracket provided in embodiment at the end of this part.
Mouse
NMRI mouse maintain Gothenburg, Sweden university rheumatology available from Scanbur BK (Sollentuna, Sweden)
In the animal factory building of system.The Goteborg Animal Experimental Ethical committee have approved the experiment.10 animals are raised in each cage, with
The Light-Dark of 12 hours is circulated, and is arbitrarily raised with standard laboratory chow and water.Test start when, animal be 6 to
16 week old.
Bacterium bacterial strain
For infection animal, S. aureus wild type bacterial strain Newman (14) and LS-1 (11) and its structure have been used
Build derivative.ClfA P are built in bacterial strain Newman336SY338A (clfAPYI) and clfAP336AY338S (clfAPYII) is derivative
Thing and being transduceed to bacterial strain LS-1 (sees below).Also use deletion mutant Newman clfA2::Tn917 mutant
DU5876 (3) and LS-1 clfA2::Tn917 mutant (J.R.Fitzgerald et al. is not delivered).Bacterium is in blood agar
Grown 48 hours on plate, harvest and be frozen in -20 DEG C containing 5% (weight/volume) BSA (Sigma Chemicals) and
In the PBS of 10% (volume/volume) dimethyl sulfoxide (DMSO).Before being expelled in animal, bacterial suspension is thawed, in PBS
Washing, and adjust to suitable cell concentration.When stimulating every time according to the culture on blood agar plate and counting Detection of colony
The number of bacterium living.
ClfAPYI and clfAPYII mutation are built in staphylococcus aureus Newman and LS-1
In this experiment, the total length ClfAA regions comprising N1, N2 and the N3 corresponding to amino acid 40-559 have been used to truncate
Thing.Be described below with accompanying drawing：
- ClfA is also known as rClfA 40-559 (SEQ ID NO 3)；
-ClfA P336SY338A is also known as clfAPYI, rclfAPY or rclfAPYI (i.e. clfAPYI40-559)
(SEQ ID NO 4)；With
-ClfA P336AY338S is also known as clfAPYII, rclfAPYII (i.e. clfAPYII 40-559) (SEQ ID
NO 5)。
P will be contained in clfA336S and Y338The PstI-BamHI fragments of the pCF77PY of the 1.02kb of A mutation
(Loughman et al., 2005) clone enters pBluescriptII SK- (Stratagene).Using HindIII by the plasmid
The pTSermC (J.Higgins is not delivered) into HindIII- cuttings is linearized and connected to produce plasmid pARM, it is temperature
Escherichia coli-staphylococcus aureus the shuttle plasmid of sensitiveness is spent, it contains P336S and Y338A replaces.
In order to reduce due to substitution P336And Y338And the control unknown risks of feature or immunoreactivity epitope are produced, we produce
Second mutant has been given birth to, wherein the order for replacing is anti-, that is, P has been produced336A and Y338S.In order to produce this, class is generated
But it is similar to pARM comprising P336A and Y338The plasmid pJH2 of S substitutions.Draw using with being used to prepare pCF77PY (6) identical flank
Thing and a pair different overlap mutant primers (mutation is represented with black matrix and underscore) carry out overlapping primers PCR to produce
pCF77PYII：
F3：GCAACTTTGACCATGGCCGCTTCTATTGACCCTGAAAATG and
R3：CATTTTCAGGGTCAATAGAAGCGGCCATGGTCAAAGTTGC
As described above, being used to produce pJH2 using the 1.02kb PstI-HindIII fragments of the plasmid --- a kind of temperature
Sensitive E. coli-staphylococcus aureus shuttle plasmid, it contains P336A and Y338S replaces.
PARM and pJH2 are transferred in RN4220 (15) by electroporation, then using bacteriophage 85 (16) by its turn
In leading staphylococcus aureus Newman (14) and LS-1 (11).In these bacterial strains, the plasmid is induced to be inserted into dye
In colour solid, then cut, will be mutated in the chromosome for staying in a certain proportion of transformant, generation Newman clfAPYI,
Newman clfAPYII, LS-1clfAPYI and LS-1clfAPYII.Loss and fibrinogen binding activity with regard to plasmid
Lose screening transformant.The integrality for passing through Southern hybridization verification clfA genes using clfA probes (data do not show).
Immunoreactive protein is verified by Westem Western blottings using the polyclonal rabbit antiserum of anti-ClfA regions A
(ClfAPY) expression (data do not show).By in the enterprising performing PCR of KpnI-BamHI fragments and product from genomic DNA
Business sequencing to mutation verify.About 700 bases and bacterial strains of the clfA genes of the bacterial strain LS-1 being sequenced
Corresponding base in the Newman clfA genes of Newman is identical.
The generation of recombinant C lfA and ClfAPY
According to former description (17), recombinant C lfA region A, the domain N123 (amino of His marks are produced from pCF40
Sour 40-559), with addition of the other polishing step by anion-exchange column.Using plasmid pCF77PY (6) as mould
Plate, clfAPYI domains N123 is cloned into pQE30 to produce pCF40PY.Using the plasmid, also by the affine layer of nickel
Analysis and anion-exchange chromatography generate recombinant C lfAPY, as the description for rClfA.Concentration and it is freeze-dried
Before, eluate is dialysed twice with PBS.
Septic arthritis and pyemia are tested
In 1-3 is tested, all of mouse (every group of n=10) is infected to trigger arthritis with bacterial strain Newman, in experiment 4
In 5, respectively with bacterial strain Newman and LS-1 infecting mouse (every group of n=10) triggering pyemia.
Experiment 1By intravenous injection 3.5 × 106The staphylococcus aureus strains Newman of cfu/ mouse or 4.3 ×
106The Newman clfAPYI mutant infecting mouses of cfu/ mouse, the two is in 200 μ l PBS.Monitoring clinical joint
Scorching and weight change is until the 7th day.Mouse is killed at the 8th day, is determined the growth of bacterium in kidney and is determined blood serum IL-6
With total IgG level.The Histological research of synovitis and osteoclasia is carried out in the joint of front and rear foot.
Experiment 2Respectively with 5.0 × 106cfu、6.0×106Cfu or 4.3 × 106The staphylococcus aureus strains of cfu
Newman, clfAPYI mutant or Newman clfA::ErmR(clfA null mutants) infecting mouse.Monitoring clinical joint
Scorching and weight change is until the 7th day.Mouse is killed at the 7th day, determines the growth of bacterium in kidney；Determine blood serum IL-6 and total
IgG levels.The Histological research of synovitis and osteoclasia is carried out in the joint of front and rear foot.
Experiment 3Respectively with 4.7 × 106cfu、3.2×106cfu、3.9×106Cfu or 4.8 × 106The golden yellow Portugal of cfu
Grape meningitidis strains Newman, clfAPYI mutant, Newman clfAPYII mutant or NewmanclfA null mutation body-sensings
Dye mouse.Monitoring clinical arthritis and weight change are until the 7th day.Mouse is killed at the 8th day, determines the life of bacterium in kidney
Long
It is closely similar to test the result of 1-3, so presenting tidal data recovering and together.
Experiment 4In, by mouse respectively with 5.2 × 107cfu、5.1×107Cfu or 3.3 × 107The golden yellow grape of cfu
Meningitidis strains Newman, clfAPYI mutant or clfA null mutants are injected intravenously.Monitoring the death rate, weight change and
Clinical arthritis are until the 10th day.
Experiment 5In, by mouse respectively with 9.4 × 106cfu、7.9×106cfu、10.7×106Cfu or 9.8 ×
106Staphylococcus aureus strains LS-1, LS-1clfAPYI mutant of cfu, LS-1 clfAPYII mutant or LS-1
ClfA null mutants infect.The monitoring death rate, clinical arthritis and weight change are until the 16th day.
Intra-articular injection bacterium
Respectively with 2.4 × 104cfu、2.4×104Cfu or 3.4 × 104The bacterial strain Newman wild types of cfu, clfAPYI dash forward
Variant or clfA knockout mutationss body (in 20 μ l PBS) every mouse knee joint of injection.Every group of N=10.At 3 days
After kill mouse, collecting knee joint is used for histopathological examination.
Vaccine inoculation is carried out with wild type and mutant recombinant C lfA
RClfA40-559, rClfAPY40-559 (i.e. rClfAPYI) for purifying or BSA are dissolved in physiological saline simultaneously
Emulsified with 1: 1 with Freund's complete adjuvant (Difco Laboratories).The 0th day by 200 μ l contain 30 μ g (=
0.53nmol) the emulsion of albumen subcutaneous (s.c.) injection.At the 11st day with the physiological saline in incomplete Freund's adjuvant
30 μ g albumen carry out first time booster immunization.Second booster immunization was carried out at the 21st day.At the 30th day by mouse bloodletting, by blood
Chilly jelly is analyzed for follow-up antibody response.
At the 31st day, with 4.0 × 106Cfu/ mouse are by being injected intravenously every group of 14-15 mouse of infection to induce purulence
Toxicity arthritis, or with 2.3 × 107Cfu/ mouse induce pyemia.Monitoring clinical arthritis, weight change and the death rate point
Not up to 11 and 15 days.Bacterial growth in determining kidney in Septic arthritis experiment.
The clinical evaluation of infecting mouse
Clinical evaluation is carried out in ignorant mode.Visually inspect each limbs.(0 is do not have for the scoring of inspection generation 0 to 3
Swelling and erythema；1 is slight swelling and/or erythema；2 is moderate swelling and/or erythema；3 is notable swelling and/or erythema).It is logical
Cross and the scoring of all four limbs of animal is added and arthritis index is obtained.Also by determining the sign i.e. weight of systemic inflammatory
Mitigate, alertness reduction and fur hair wrinkle to check every overall state of mouse.In the case of severe sepsis, when
Judge certain mouse it is sick it is too serious so that it is living only 24 hours when, killed by disconnected cervical approach and be considered due to
Pyemia and death.
Histological examination
The histological examination in joint is carried out using the modification (8) of the previously described method (18).
The bacteriology checking of the kidney of infection
Aseptic separation kidney, be placed on ice, homogenate, serial dilution and coat on blood agar plate in PBS.At 37 DEG C
Incubate 24 hours afterwards, determine the cfu numbers of each pair kidney.
The measure of serum IgG
Total IgG level in serum is determined by radial immunodiffusion technique (19).Goat anti-mouse IgG and mouse
IgG reference materials are purchased from Southern Biotech, Birmingham, AL.
Specific antibody-ELISA
Blood serum sample is obtained from through immune mouse within 9 days after second booster immunization.Determined by ELISA and be directed to
The serum specific antibody reaction of rClfA and rClfAPY.With recombinant protein coating microplate (the 96- holes of 5 μ g/ml in PBS；
Nunc).Closed reagent, blood serum sample, biotinylated antibody and ExtrAvidin- peroxidase dilute in PBS.Root
Description (8) preceding according to this carries out the inspection.All of blood serum sample is anti-with the absorbance monitoring at 405nm with 1: 20000 dilution
Precursor reactant.
In being taken turns second, in order to the more accurate of specific antibody reaction determines in obtaining different immune groups, in several blood
Reaction is determined in clear dilution factor.Therefore, all of blood serum sample dilutes with 1: 5000,1: 20000,1: 80000 and 1: 320000,
Antibody response is monitored with the absorbance at 405nm.
IL-6 is analyzed
Blood serum IL-6 is detected according to the previously described method (20).
Statistical analysis
Being checked using Mann-Whitney U carries out statistical evaluation.P ＜ 0.05 are considered as conspicuousness.Unless otherwise
Indicate, otherwise data report be median, interquartile range and 80% middle ware away from.
As a result
The replacement that ClfA is combined required two amino acid with fibrinogen hinders septic arthritis and pyemia
Generation
Exchanged by allele will be known as two amino acid necessary to ClfA is combined with fibrinogen (P336 and
Y338) change with the mutant of producing bacterial strain Newman and LS1, it is in cell surface expression non-fibrous protein original associativity ClfA
Albumen.Detect the expression and integrality of the albumen by western blot, it was demonstrated that the mutain is in bacterium surface table
It is correct size up to albumen that is good and expressing.
Have studied Newman wild types and Newman clfA P336S Y338A (clfAPYI) triggers septic arthritis
Ability.3.5 × 10 are inoculated with respectively by intravenous6To 5.0 × 106CFU (cfu) and 3.2 × 106To 6.0 ×
106The Newman wild types and clfAPYI mutation induction septic arthritis of cfu.To arthritic development clinical research 7
My god.During whole experiment, compared to significantly lower (the P ＞ of severity of arthritis that wild-type strain, clfAPYI mutant trigger
0.001, Figure 1A).In most time point, for Newman clfAPYI, arthritic frequency is relatively low (Fig. 6).
It is surprising that the new amino acid composition in ClfAPYI molecules seems to be adapted to be defendd with the antibacterium of host
Interact.In order to check this possibility, a kind of new construct is prepared for, wherein replacing P336 with different amino acid
With Y338 (clfA P336A Y338S：clfAPYII).With 3.9 × 106What the mouse of the NewmanclfAPYII inoculations of cfu produced
Arthritis degree is low as clfAPYI mutant (Figure 1A), and with similar frequency (Fig. 6).The strong table of this result
Bright, the reduction of arthritis level is facilitated in the forfeiture that fibrinogen is combined.
ClfA may participate in arthritic generation by the mechanism for not being related to fibrinogen to combine.May to test this
Property, the ClfA that will lack ClfA albumen deletes the mutation of mutant and the non-fibrous protein original associativity ClfA albumen of expression modification
Body is compared.But, with 4.3 × 106To 4.8 × 106The arthritis that the mouse of the clfA null mutants infection of cfu produces
The mouse infected with clfAPYI and clfAPYII mutant does not have difference (Figure 1A) in mode.Arthritic frequency is also not
Differentiable (Fig. 6).
Histological research also has been carried out to the joint infected.In experiment 1 and 2, compared to the mouse that wild type infects,
Synovitis in the mouse of Newman clfAPYI infection is significantly relatively light (respectively P=0.02 and 0.001).In Newman
In the sample of clfAPYI infection, (osteoclasia is that the main of mankind's septic arthritis sequelae is lured to there's almost no osteoclasia
Cause) (experiment 2, P=0.001).Compared with the mouse of Newman wild types infection, the induction of Newman clfA null mutants
Synovitis and osteoclasia are relatively light (respectively P=0.003 and 0.006), but to a certain extent than Newman clfAPYI
Group is serious, although be not very notable.
Next the metabolic consequence that clfA mutation are caused for course of infection is analyzed.Infected with Newman wild-type strains
Mouse in experimentation Body weight loss be up to about 30%.With the mutant Newman of fibrinogen binding deficient
Almost without any reduction, (P ＞ 0.0001 are relative to wild for the mice weights of clfAPYI and Newman clfAPYII infection
Type).Conversely, Newman clfA null mutants have medium effect for weight loss, cause significantly less than wild-type bacteria
Strain, but it is significantly higher than clfAPYI and clfAPYII mutants which hads (P≤0.02, Figure 1B as a rule).
The IL-6 as the measurement of systemic inflammatory reaction is analyzed within 7-8 days the of infection.The table of IL-6
Expression patterns are similar to weight change.Newman wild types trigger high-caliber blood serum IL-6 (4.8 (2.8,5.7) ng/ml),
Newman clfAPYI mutant triggers significantly lower IL-6 (0.2 (0.07,2.4) ng/ml, P ＜ 0.0001) and Newman
ClfA null mutants produce medium reaction (2.5 (1.3,3.2) ng/ml), and it is equal with wild type and clfAPYI mutant groups
There were significant differences (respectively P=0.009 and P=0.008) (median, interquartile range).
Compared with both Newman clfAPY mutant and Newman clfA null mutants, in Newman wild types
In the mouse of infection, significantly higher (the respectively P ＜ 0.0001, P=0.011, and P=0.005 of bacterial growth in kidney；Figure
2).Compared to the mouse that Newman clfAPYI- infect, in the kidney of the mouse of Newman clfA null mutants-infection
Bacterial growth is significantly higher (P=0.0005, Fig. 2).
Total IgG in determining mice serum in 7-8 days the of infection.Newman clfAPYI- and NewmanclfA without
The increase for imitating the IgG levels in two groups of mutant-infection is substantially lower than with the mouse of wild-type strain infection (respectively
3.1 (1.2,4.9)；2.3 (1.0,2.6)；With 6.4 (5.0,11.0) (median, interquartile range)；P≤0.0003).
There is no significant difference between two mutant groups.
The death rate is 17% in the mouse of Newman wild types infection, in Newman clfAPYI and clfAPYII mutation
It is 0% in body group, is 30% in Newman clfA null mutant groups.Between wild type and clfAPYI groups and
There is significant difference (respectively P ＜ 0.05 and P ＜ 0.01) in the death rate between clfAPYI and clfA null mutant groups.
In the mouse for having infected the staphylococcus aureus of ClfA of expression fibrinogen binding deficient, general is scorching
The directly or indirectly sign of disease seems relatively low.It was unexpectedly determined that not expressing the bacterial strain of ClfA all induction of than expression ClfAPY
The stronger systemic inflammatory of the bacterial strain of mutant.
By increasing the dosage of inoculation of staphylococcus aureus induction of pyemia.With 5.2 × 107The Newman of cfu is wild
Raw type, 5.1 × 107The Newman clfAPYI mutant of cfu and 3.3 × 107The Newman clfA null mutants injection of cfu
Mouse.In 5 days, all of mouse for having infected wild type is all dead, but after injection 10 days, 10 injections
Only has 1 death (P ＜ 0.0001, Fig. 3) in the mouse of clfAPYI mutant.The mouse of clfA null mutants is injected
Life span is also considerably shorter than the mouse (P ＜ 0.0001, Fig. 3) of clfAPYI mutant infection.In this experiment, with clfA without
The mouse that effect mutant stimulates produces the arthritis for being significantly more than clfAPYI mutant groups, while they lose significantly more
Weight (Fig. 7 and 8).Therefore, by with Septic arthritis experiment in systemic inflammatory the analogy of measurement phase find, if
ClfA molecules are expressed, then the existence of mouse is extended, as long as the ClfA molecules do not have fibrinogen binding property.
Bacterial injections are entered into joint
In order to determine whether the inflammatory reaction in joint depends on fibrinogen to combine, by Newman wild types,
Newman clfAPYI or Newman clfA blank direct injection enters the knee joint of mouse, so as to bypass general compartment.3
Synovitis is studied by Histological method after it, including the polymorphonuclear of articular cavity permeates, and osteoclasia.Mouse is in a knee
Place receives 2.4 × 104The wild type of cfu, 2.4 × 104The clfA null mutants of cfu or 3.4 × 104The clfAPYI of cfu dashes forward
Variant.For the knee for having infected wild type, the Histological Index of synovitis and the polymorphonuclear infiltration in articular cavity for 0.25 (0,
3.0) it is, 2.38 (0.25,3.0) for clfA null mutants, is 0.25 (0,0.25) (middle position for clfAPYI mutant
Number, interquartile range).For wild type, the Histological Index of osteoclasia is 0 (0,1.0), is for clfA null mutants
1.0 (0,1.0), are 0 (0,0) (median, interquartile range for clfAPYI mutant；In clfAPYI mutant and
P=0.01 between clfA null mutants).Because clfAPYI mutant triggers few synovitis and destruction, so, although
The fact that in the presence of " give the amount of the bacterial strain of mouse 40% more than other bacterial strains ", but still draw a conclusion：ClfA promotes
Fibrinogen to combine for IA maximum inflammatory reaction be required.Again, combined relative to fibrinogen and lacked
Sunken ClfA mutant, the missing of ClfA expression enhances inflammation.
PY mutation in bacterial strain LS-1
In order to determine whether ability that ClfA is combined with fibrinogen influences the poison of other staphylococcus aureus strains
Power, by the staphylococcus aureus strains LS-1 of clfAPYI, clfAPYII and clfA null mutation transduction to expression TSST-1.
With 9.4 × 106LS-1 wild types, 7.9 × 10 of cfu6LS-1 clfAPYI, 10.7 × 10 of cfu6The LS-1 of cfu
ClfAPYII or 9.4 × 106The LS-1 clfA null mutants of cfu stimulate mouse.Septicopyemia is studied by monitoring survival rate
Disease.At 16 days afterwards, the mouse only 40% for being stimulated with wild-type strain is survival, and with clfAPYI mutant and clfA
It is survival that the mouse that null mutant group stimulates has 90%, and it is survival that the mouse infected with clfAPYII mutant has 80%
(Fig. 4).The clfAPYI mutant of LS-1 and the virulence of clfA null mutants significantly lower (respectively P=0.014, P=
0.05 and P=0.03).
It is immunized with recombinant C lfA albumen
Restructuring wild type ClfA A domain proteins are all have studied in septic arthritis model and sepsis model
And mutant ClfAPYI albumen (rClfAPY) carries out the effect of vaccine inoculation (rClfA).By mouse sensitization, then compareing egg
White BSA, rClfA or rClfAPY carry out booster immunization twice, then with 4.0 × 106The staphylococcus aureus strains of cfu
Newman is infected with triggering septic arthritis, or with 2.3 × 107The bacterial strain Newman of cfu infects to induce pyemia.With
Control mice is compared, and immune septic death is generated with what rClfAPY (i.e. ClfAPYI recombinant protein As domain) was carried out
Conspicuousness protects (P=0.01, Fig. 5), and rClfA is immune without generation conspicuousness protection.1 day before bacterium infection,
In rClfAPY immune mouse, the Specific serum antibodies reaction (A for rClfAPY and rClfA405=0.39 (0.33,
0.56) and 0.71 (0.52,0.81)) than rClfA be immunized the much higher (A of mouse405=0.13 (0.07,0.17) and 0.15
(0.10,0.24), P ＜ 0.0001 (median, interquartile range) in being contrasted at two).Immune animal only has for control
Background level (A405nm=0 and 0.01 (0,0.01) (median, interquartile range)).With relatively low arthritis bacterium dosage sense
The immune mouse of dye to wherein generate the similar antibody response for rClfA and rClfAPY induction of pyemic mouse
(data do not show).It is respectively provided with for there is arthritic protection so that rClfA and rClfAPY is immune, although this protection is not
(Fig. 9) of conspicuousness.
After infection in the 5th to 9 day, compared with control mice, weight is damaged in the mouse that rClfAPY and rClfA is immunized
Mistake is substantially reduced (data do not show).
It was observed that the 11st day after infection, the reduction of bacterial growth in the kidney of the mouse being immunized with rClfAPY and rClfA
Trend (BSA：38 (3,436)；rClfAPY：7 (2,17)；rClfA：10 (7,54) × 107Cfu/ is to kidney).
Determined to obtain the more accurate of specific antibody reaction in different immune groups, in several serum dilutions (the
Two wheel) in reaction is determined.Data display：In the mouse infected with septic and arthritis bacterium dosage, and from rClfA
The serum of the mouse of wild-type immune is compared, the specific antibody of the serum from rClfAPY immune mouses for rClfAPY and
The potency of rClfA wild type antigens is all higher because, in all of comparing, with rClfAPY be immunized mouse each
Antibody response in serum dilution according to absorbance measurement is all significantly higher (P ＜ 0.0001 to P=0.008, Figure 12-15).
BSA is immune only to have triggered background antibody reaction.
Conclusion
Result shows strongly：ClfA- fibrinogens interact most important for bacterial virulence and disease consequence.
Cause the ability aspect of septic death, ClfA is related with enhanced virulence to the ability that fibrinogen is combined.What is surveyed
In two aureus strains, the septic death of clfAPY mutation inductions is below wild type.Equally, non-fibre is being infected
In the mouse of fibrillarin original associativity clfAPY mutant, arthritic seriousness is significantly reduced.
It is in thermophilic that fibrinogen-bacterial cell surface interacts for a possible mechanism of the promotion of virulence
The suppression (5) of the phagocytosis of property granulocyte.Host defense of the neutrophil cell for infection of staphylococcus aureus early stage
It is critical that (13).If without neutrophil cell, the bacterial growth in blood and kidney will expand strongly, arthritis
Frequency and the death rate increase.The phagocytosis for neutrophil cell that the fibrinogen carried out by ClfA is mediated
Suppression can at least partly explain stronger virulence of the wild-type S. aureus bacterium relative to clfAPY mutant.Fiber
The combination of proteinogen and ClfA can by reduce opsonin deposit or by reduce neutrophil cell acceptor with it is opsonic
Contact and reduce the opsonophagocytosis of neutrophil cell.Alternatively, with reference to fibrinogen may block unknown protectiveness
Host factor and the combination of staphylococcus aureus.Bacterium is promoted to lead to another possibility is that fibrinogen-ClfA interacts
Cross blood vessel and enter tissue or promotion cluster in the tissue.
It was unexpectedly determined that our data are also shown that virulence of the ClfA null mutants than clfAPY mutants which had
It is stronger.ClfA albumen is possible to the function of having in addition to being interacted with fibrinogen in vivo.As shown in this research,
This interaction is clearly unfavorable for host.Other functions of ClfA are failed to understand so far, but ClfA is showed
Non-fibrous protein original the hematoblastic aggregation of dependence may cause circulation in a large amount of staphylococcus aureuses capture, and then
Bacterium-platelet complex is removed by reticulo-endothelial system.Will be easy in wild type and clfAPY mutant strains
There is the staphylococcic removing of this platelet aggregation mediation in ground, but will not then occur in clfA knocks out body.Although
Fibrinogen interacts and will cover other events in wild-type strain, but such bacterium is clear in clfAPY mutant
Except may be highly profitable for host.
ClfA knockout mutationss body is in the degree of protection and staphylococcus aureus strains LS-1 of septic death
ClfAPY mutant is identical, but protects less in bacterial strain Newman, if having protected.ClfA expression for
The general impacts of bacterial virulence can be different between different staphylococcus aureus strains, and this depends on other virulence
The expression of the factor and presence.
It is abundant in view of the fibrinogen in the synovia of inflammation and fiber protein content, on " clfAPY mutant exists
Whether same or relatively low virulence is shown in articular cavity " problem be have certain significance.Our tables of data
Bright, clfAPY mutant is destructive relatively low for cartilage and bone.
Recombinant C lfA A domains non-fibrous protein original associativity P336Y338The protective effect of mutant is than wild type rClfA
It is higher.With ClfAPY be immunized and be very likely to the more preferable immune response of induction, because being directed to immunogene and wild type ClfA eggs
Bai Jun has triggered specific antibody higher to react.Importantly, it produces bigger resistance purulence than wild type ClfA inductions
The protective immunological reaction of toxic death.
In a word, our result indicate that rClfAPY is than the wild type recombinant C more preferable candidate vaccines of lfA.We assume that：
Important epitope in due to ClfA molecules is hidden, thus during immune wild type ClfA albumen and fibrinogen combination
Antigen presentation is reduced, therefore destroys the generation of specific antibody.
Embodiment 2
RClfA a-quadrants truncate (rClfA 221-559) comprising N2 and N3
Material and method
The program shown in embodiment 1 is continued to use in the present embodiment, is which used
- rClfA 221-559 (i.e. the ClfA a-quadrants truncate comprising the N2 and N3 corresponding to amino acid 220-559)
-rClfAPY221-559；With
-BSA。
Every group has 15 female NMRI mouse, and when beginning is tested, it is 8 week old.In the present embodiment, for be immunized
Construct is ClfA wild types/ortho states N2N3 truncates, the ClfA N2N3 truncations with the mutation PY as defined in embodiment 1
Thing.BSA is with comparing.
Vaccine inoculation is carried out with wild type and mutant recombinant C lfA
Program according to embodiment 1 is immunized with rClfA 221-559, rClfAPY 221-559 or BSA to mouse.
RClfA221-559, the rClfAPY221-559 (i.e. ClfAPYI recombinant protein As subdomain N2 and N3) that will be purified
Or BSA is dissolved in PBS and is emulsified with 1: 1 with Freund's complete adjuvant.The 0th day by 200 μ l contain 30 μ g (=
0.79nmol) emulsion of albumen passes through hypodermic injection.At the 12nd day with 30 μ in the physiological saline in incomplete Freund's adjuvant
G albumen carries out first time booster immunization.Second booster immunization was carried out at the 22nd day.At the 31st day by mouse bloodletting, by serum
Freeze and analyzed for follow-up antibody response.
Specific antibody-ELISA
Blood serum sample is obtained from immune mouse within 9 days after second booster immunization.Determined by ELISA and be directed to
The serum specific antibody reaction of rClfA221-559 and rClfAPY221-559.With the recombinant protein bag of 5 μ g/ml in PBS
By microplate (96- holes；Nunc).Closed reagent, blood serum sample, biotinylated antibody and ExtrAvidin- peroxidase are equal
Diluted in PBS.The inspection was carried out according to former description (8).All of blood serum sample is with 1: 5000,1: 20000,1:
80000 and 1: 320000 is diluted, and antibody response is monitored with the absorbance at 405nm.
As a result
Specific antibody reacts
According to embodiment 1, absorbance measurement antibody response is passed through in ELISA inspections with 4 different serum dilutions.
The data of acquisition are closely similar with data in embodiment 1.
It was found that immune relative to what is carried out with ortho states rClfA221-599, with rClfAPY221-559 carry out it is immune very
There may be the specific antibody for ortho states rClfA221-559 and rClfAPY221-559 of more efficient valency, because except one
Beyond individual, in all of contrast, in each serum dilution, immune mouse is carried out all with aobvious with rClfAPY221-559
Write the antibody response with absorbance measurement higher and (P=0.001 to 0.025, see Figure 16 and 17).BSA is immune only to trigger background
The antibody response of level.
Conclusion
It was found that the immune generation carried out with rClfAPY221-559 albumen for immunogene and wild type ClfA eggs
White antibody response is all significantly higher than with being immunized that ortho states albumen is carried out.
Based on these discoveries, we conclude that：Either comprising the amino acid 40-550 or implementation in embodiment 1
PY albumen comprising amino acid 221 to 559 in example 2, all triggers more immune than what correspondingly sized ortho states ClfA was carried out so that PY- is immune
More preferable immune response.
Embodiment 3
ClfA a-quadrants truncate (δ/Δ locking truncate)
Material and method
The program shown in embodiment 1 is continued to use in the present embodiment, which uses following construct
- rClfA 221-531 (i.e. comprising N2 and N3 amino acid 220-559 but without locking peptide ammino acid 532-538 and with
The rClfA a-quadrants truncate of Pro-rich residue afterwards).
Every group has 15 female NMRI mouse, and when beginning is tested, it is 8 week old.In the present embodiment, above-mentioned structure is used
Body is built for being immunized.Program in embodiment 1 is with above-mentioned truncate immune mouse.
Vaccine inoculation is carried out with wild type and mutant recombinant C lfA
The rClfA221-531 of purifying is dissolved in PBS and is emulsified with 1: 1 with Freund's complete adjuvant.At the 0th day
The emulsion that 200 μ l contain 0.79nmol albumen is passed through into hypodermic injection.At the 12nd day with the physiology salt in incomplete Freund's adjuvant
0.79nmol albumen in water carries out first time booster immunization.Second booster immunization was carried out at the 22nd day.Will be small at the 31st day
Mouse bloodletting, serum is freezed and is analyzed for follow-up antibody response.
Specific antibody-ELISA
Blood serum sample is obtained from immune mouse within 9 days after second booster immunization.Specific antibody is determined by ELISA
Serum levels.With rClfA221-531 albumen coating microplate (the 96- holes of 4.6 μ g/ml；Nunc), the protein content and implementation
The rClfA221-559 and rClfAPY221-559 of 5 μ g/ml in example 1 and 2 are equimolar amounts.Closed reagent, blood serum sample,
Biotinylated antibody and ExtrAvidin- peroxidase dilute in PBS.The inspection was carried out according to former description (8)
Test.All of blood serum sample is diluted with 1: 5000,1: 20000,1: 80000 and 1: 320000, with the absorbance at 405nm
Monitoring antibody response.
As a result
According to embodiment 1, the absorbance measurement antibody response in being checked with ELISA-.It was found that being carried out with rClfA221-531
Immune generation immune response, as specific antibody reaction survey (Figure 18).
Conclusion
It was found that rClfA221-531 has an effect as immunogene, because the antigen triggers specific antibody reaction.
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Claims (16)
1. restructuring staphylococcus clumping factor A (ClfA) of the amino acid residue 221-531 comprising fibrinogen calmodulin binding domain CaM
Or purposes of its fragment in the medicine for being used for treating or prevent microorganism infection is prepared, the restructuring staphylococcus clumping factor
A (ClfA) or its fragment include at least a portion fibrinogen calmodulin binding domain CaM and stimulate exempt from bigger than wild type ClfA albumen
Epidemic disease is reacted, and is replaced with least one amino acid residue at amino acid residue Pro336 or Tyr338, is reduced with producing to have
Or without the recombinant fibrinogen associated proteins with fibrinogen Non-covalent binding ability.
2. restructuring staphylococcus clumping factor A (ClfA) of claim 1 or the purposes of its fragment, the restructuring staphylococcus gather
Collection factors A (ClfA) or its fragment replace at amino acid residue Pro336 and Tyr338 including amino acid residue.
3. restructuring staphylococcus clumping factor A (ClfA) of claim 1 or the purposes of its fragment, the restructuring staphylococcus gather
Collection factors A (ClfA) or its fragment replace at amino acid residue Tyr256, Lys389, Glu526 and Val527 or with Ser
Include at least one at the amino acid residue Ala254 of Ala or at the amino acid residue Ile387 for replacing Ile with Ala or Ser
Other amino acid residue substitution.
4. restructuring staphylococcus clumping factor A (ClfA) of claim 1 or the purposes of its fragment, the restructuring staphylococcus gather
Collection factors A (ClfA) or its fragment are in amino acid residue Tyr256, Lys389 or in the amino acid residue with Ser substitutions Ala
Include at least one other amino acid residue at Ala254 or at the amino acid residue Ile387 for replacing Ile with Ala or Ser
Substitution.
5. restructuring staphylococcus clumping factor A (ClfA) of claim 1 or the purposes of its fragment, the restructuring staphylococcus gather
The amino acid residue 221 to 559 of collection factors A (ClfA) or its fragment comprising fibrinogen land.
6. restructuring staphylococcus clumping factor A (ClfA) of claim 1 or the purposes of its fragment, wherein restructuring staphylococcus gathers
Collection factors A (ClfA) is reduced or is prevented with fibrinogen by the Non-covalent binding that depressed place, lock and locking occur.
7. the purposes of restructuring staphylococcus clumping factor A (ClfA) of any one of preceding claims, the restructuring grape ball
Fibrinogen calmodulin binding domain CaM A of bacterium clumping factor A (ClfA) comprising fibrinogen binding protein, the wherein substitution of amino acid
The noncovalent interaction with fibrinogen is reduced, it is described to dredge preferably by preventing or reducing the combination of part and hydrophobic pocket
The subprovince domain N2 and N3 of the protein-bonded region A of water bag septate fibre proteinogen.
8. the purposes of restructuring staphylococcus clumping factor A (ClfA) of any one of claim 1-6, wherein ClfA fibrins
There is the substitution of at least one or more nucleic acid or amino acid in the hydrophobic residue of former calmodulin binding domain CaM.
9. the purposes of restructuring staphylococcus clumping factor A (ClfA) of any one of claim 1-6, wherein amino acid residue
Ala254, Tyr256, Pro336, Tyr338, Ile387, Lys389, Glu526 and/or Val527 are replaced by Ala or Ser.
10. the purposes of restructuring staphylococcus clumping factor A (ClfA) of any one of claim 1-6, wherein ClfA fibers egg
The residue P of white original calmodulin binding domain CaM336And/or Y338Replace to produce rClfAP by serine or alanine336S Y338A or
rClfAP336A Y338S。
The purposes of restructuring staphylococcus clumping factor A (ClfA) of any one of 11. claim 1-6, wherein restructuring grape ball
Bacterium clumping factor A (ClfA) has according to any SEQ ID NO:1 to 3 amino acid sequence or its fragment, wherein residue P336
And/or Y338Replaced by serine and/or alanine, or comprising any SEQ ID NO:4th, 5,7,8,11,12 and 14 amino acid
Sequence.
The purposes of restructuring staphylococcus clumping factor A (ClfA) of any one of 12. claim 1-6, the restructuring grape ball
Bacterium clumping factor A (ClfA) is included：
Subprovince domain N123, across the amino acid residue 40-559 of fibrinogen calmodulin binding domain CaM.
The purposes of restructuring staphylococcus clumping factor A (ClfA) of any one of 13. claims 1 to 12, wherein micro- life
Thing infection is caused by staphylococcus.
The purposes of restructuring staphylococcus clumping factor A (ClfA) of 14. claims 13, wherein the staphylococcus is golden yellow
Staphylococcus.
15. include restructuring staphylococcus clumping factor A (ClfA) defined in claim any one of 1-12 or the epidemic disease of its fragment
Seedling.
16. comprising restructuring staphylococcus clumping factor A (ClfA) defined in claim any one of 1-12 or its fragment and
The Immunogenic agents composition of pharmaceutically acceptable adjuvant.
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| EA019516B1 (en) | 2008-01-31 | 2014-04-30 | Де Провост, Феллоус Энд Сколарс Оф Де Колледж Оф Де Хоули Энд Андивайдид Тринити Оф Куин Элизабет, Неар Дублин | Treatment of microbial infections |
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| EP2488547B1 (en) | 2009-10-15 | 2017-12-06 | University Court of the University of Edinburgh | Staphylococcal antigens |
| CN101843899B (en) * | 2010-05-24 | 2012-11-07 | 中国人民解放军第三军医大学 | Methicillin-resistant staphylococcus aureus (MRSA) recombinant multivalent subunit genetic engineering vaccine and method for preparing same |
| US9095540B2 (en) | 2010-09-09 | 2015-08-04 | The University Of Chicago | Methods and compositions involving protective staphylococcal antigens |
| WO2013159021A1 (en) * | 2012-04-20 | 2013-10-24 | The Texas A&M University System | Engineered collagen binding mscramm with enhanced affinity for collagen |
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| US9238090B1 (en) | 2014-12-24 | 2016-01-19 | Fettech, Llc | Tissue-based compositions |
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